Urothelial carcinoma (UC) of the bladder is the seventh most common malignant tumor worldwide among men, with an approximate number of 350.000 new cases diagnosed per year. UC is the fourth cause of death due to neoplasm and is the second most common tumor of the urinary system after prostate cancer and with it, due to its high relapse rate, is the main cause of uro-oncological attention (Boyle P., Ferlay J. Cancer incidence and mortality in Europe, 2004. Ann Oncol 2005; 16:481-8).
More than 90% of cases of UC of the bladder are transitional cell carcinomas, although they can also present as adenocarcinomas and squamous cell carcinomas. The transitional cell epithelium is located in the inner layer of the urothelial mucosa and is separated from the muscle layer by the lamina propria. Approximately 75-80% of the cases of UC are superficial tumors, i.e., they do not invade the muscle layer, so they are called non muscle-invasive bladder cancer (NMIBC). 70-80% of them are confined only to the urothelial mucosa (Ta and CIS stage), whereas 20-30% reach the lamina propria (T1 stage) without invading the muscle layer of the bladder wall (Cheng L, Weaver A L, Neumann R M, Scherer B G, Bostwick D G. Substaging of T1 bladder carcinoma based on the depth of invasion as measured by micrometer: A new proposal. Cancer 1999; 86:1035-43). NMIBCs are characterized by their multiplicity, their tendency to relapse and their good prognosis. The survival rate of NMIBC patients is 90% at 5 years and 80% at 10 years (Pansadoro V, Emilliozzi P, Defidio L, et al. Bacillus Calmette-Guerin in the treatment of stage T1 grade 3 transitional cell carcinoma of the bladder: long-term results. J Urol 1995; 154:2054-8). While the risk of recurrence in these patients is approximately 70%, only 10-15% progress to muscle-invasive bladder cancer (MIBC). A close association between the tumor grade and the risk of tumor progression has been observed (Jordan A M, Weingarten J, Murphy W M. Transitional cell neoplasms of the urinary bladder. Can biologic potential be predicted from histologic grading? Cancer 1987; 60:2766-74). Most Ta tumors are low grade tumors. These tumors frequently return but less than 5% progress. On the other hand, many T1 tumors are high grade tumors and 30-50% progress to muscle layer-infiltrating tumors. In contrast, approximately 20% of the cases of UC present as MIBC at the time of diagnosis. These are aggressive tumors that lead to death in 2 years if they are not treated. 50% of MIBC patients die after 2 years due to distant metastasis or local relapse despite receiving radical surgical treatment. The basic problem of MIBC lies in the brevity of its pre-invasive step, which conditions that when the clinical phase is reached, 27-60% of the cases have already affected the deep muscle layer, 25% reach the prevesical fat and 14% have clinically detectable distant metastases.
Current diagnostic systems are based on a combination of urinary cytology (from desquamated cells in the urine) and the direct observation of the bladder by means of cystoscopy. Cystoscopy is in fact the main tumor diagnostic and follow-up technique. It is performed by transurethral route, so it is an invasive and rather bothersome technique for patients. The sensitivity and specificity of this technique were thought to be rather high, although improvements in the technique itself (fluorescent cystoscopy) indicate that this is probably not the case and that part of the recurrence observed in superficial tumors could be due to the lack of total resection of non-visible parts thereof (Jones J S. DNA-based molecular cytology for bladder cancer surveillance. Urology 2006; 67:35-45). Furthermore, the interpretation of the cytology is highly dependent on the observer, therefore there can be inter-observer differences, especially in low grade tumors.
Advancement in knowledge about molecular events leading to UC progression has stimulated the study of gene expression profiling by means of DNA microarrays using RNA obtained from different classes of UC, including non-muscle and muscle-invasive tumors (Bastacky S, Ibrahim S, Wilczynski S P, Murphy W M. The accuracy of urinary cytology in daily practice. Cancer 1999; 87:118-28; aboe M, Marcussen N, Jensen K M, Thykjaer T, Dyrskjot L, Orntoft T F. Gene expression profiling of noninvasive primary urothelial tumours using microarrays. Br J Cancer 2005; 93:1182-90; Thykjaer T, Workman C, Kruhoffer M, et al. Identification of gene expression patterns in superficial and invasive human bladder cancer. Cancer Res 2001; 61:2492-9), different stages of UC progression (Sanchez-Carbayo M, Socci N D, Charytonowicz E, et al. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes. Cancer Res 2002; 62:6973-80) and in patients with a different clinical progression (Sanchez-Carbayo M, Socci N D, Lozano J, Saint F, Cordon-Cardo C. Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays. J Clin Oncol 2006; 24:778-89). The same approach has also been used for the identification of a panel of 14 predictive genes which separate UC-responding patients from non UC-responding patients with respect to different therapeutic agents (Takata R, Katagiri T, Kanehira M, et al. Predicting response to methotrexate, vinblastine, doxorubicin, and cisplatin neoadjuvant chemotherapy for bladder cancers through genome-wide gene expression profiling. Clin Cancer Res 2005; 11:2625-36).
Although direct urothelial tissue analysis is the most comfortable alternative, to develop a routine diagnostic method it would be of great interest that it is not invasive because invasive maneuvers reduce the patients' quality of life and represent a much larger economic health burden. Blood, and particularly urine which is in contact with the entire bladder epithelium, and therefore with the tumor mass, are suitable sources of biological material for the detection of tumor markers, given that they represent an easy and non-invasive way of obtaining the sample to be analyzed.
A considerable number of papers have focused on studying tumor markers in urine in search of a non-invasive diagnostic method for diagnosing UC of the bladder. In fact, different tests have been marketed for this purpose (NMP22, UroVysion, ImmunoCyt, Accu-Dx, etc.), but even though most of them are more sensitive than urinary cytology, the latter is still the most specific.
The identification of marker genes for bladder cancer is complex due to the heterogeneous nature of said tumors. Diagnostic methods comprising the detection of marker proteins in urine (WO 2008/119858A1) have recently been developed, though it is only useful in the diagnosis of transitional carcinoma. International patent application WO 2008/113870 describes, among others, an in vitro non-invasive method for the diagnosis and/or prognosis of bladder cancer based on the expression profile of the ANXA10, C14orf78 (AHNAK2), CTSE, CRH, IGF2, KLF9, KRT20, MAGEA3, POSTN, PPP1R14D, SLC1A6, TERT, ASAM and MCM10 genes in a bladder fluid sample; although this method has high sensitivity and specificity, its use requires analyzing 14 markers, which increases the cost of the analysis and makes it very complex from a methodological viewpoint.
Despite there being some non-invasive diagnostic methods for diagnosing bladder cancer, none of them is being used routinely in clinical practice, so there is still a need to develop alternative methods for the non-invasive diagnosis of bladder cancer which allow diagnosing bladder cancer with high sensitivity and specificity and require the analysis of a smaller number of markers.